Daunorubicins/Doxorubicins
Topoisomerase I

A sulforhodamine B assay demonstrates the cytotoxic effects of PNU-159682 (0-500 nM; exposed to the compounds for 1 hour and then cultured in compound-free medium for 72 hours). This was observed in human tumor cell lines. For the cells HT-29, A2780, DU145, EM-2, Jurkat, and CEM, the IC70 values are 0.577 nM, 0.39 nM, 0.128 nM, and 0.081 nM, 0.086 nM, and 0.075 nM, respectively[1]. It works against human tumor cell lines, with IC70 values for MMDX and doxorubicin ranging from 68 nM to 578 nM and 181 nM to 1717 nM, respectively[1]. MMAE is not as effective against NHL cell lines as PNU-159682. In an assay for cell viability, PNU-159682 is antagonistic to BJAB. Luc, WSU-DLCL2, SuDHL4.Luc, Granta-519, and Luc, with corresponding IC50 values of 0.10 nM, 0.020 nM, 0.055 nM, and 0.1 nM. However, MMAE opposes BJAB. Luc, WSU-DLCL2, Granta-519, SuDHL4.Luc, and 0.54 nM, 0.25 nM, 1.19 nM, and 0.25 nM, in that order[2].
PNU-159682 has the potential to create a new class of ADCs and is thousands of times more cytotoxic than doxorubicin. In vitro, PNU159682?to?anti-CD22?antibody (anti-CD22-NMS249) demonstrates potent anti-tumor properties. In vitro viability assays of NHL cell lines, anti-CD22-NMS249 (PNU159682-to-anti-CD22 antibody) is active and 2–20 times more potent than pinatuzumab vedotin; the ADC anti-CD22-NMS249 is against BJAB. The IC50 values of Luc, Granta-519, SuDHL4.Luc, and WSU-DLCL2 are 0.058 nM, 0.030 nM, 0.0221 nM, and 0.01 nM, in that order[3].
The activity of PNU-159682 (100 μM) to inhibit topoisomerase II unknotting is weak. With an IC50 of 25 nM, PNU-159682 exhibits a cytotoxic effect on SKRC-52 cells that express CAIX[4].

In the murine L1210 leukemia model, PNU-159682 (single-dose; intravenous; 15 μg/kg) is the maximum tolerated dose. PNU-159682 exhibits enhanced in vivo antitumor activity. PNU-159682's antitumor effect (life span increase of 29%) is similar to that of 90 μg/kg MMDX (life span increase of 36%)[1].
In MX-1 human mammary carcinoma mice, PNU-159682 (i.v. 4 μg/kg; q7dx3; 40 days) exhibits a therapeutic response. Furthermore, four of the seven mice given PNU-159682 show total tumor regression starting on day 39[1].
PNU-159682 can be used to create a new class of ADCs because it is more cytotoxic than doxorubicin. In vivo, PNU159682?to?anti-CD22?antibody (anti-CD22-NMS249) demonstrates potent anti-tumor properties. Mice respond well to the ADC dose (anti-CD22-NMS249; 50 μg/m2 conjugated PNU-159682), which causes less than 10% weight loss[2].
Anti-CD22-NMS249 (single dose; 2 mg/kg) has comparable efficacy to anti-CD22-vc-MMAE in the BJAB.Luc model. AntiCD22-NMS249, at a dose of 2 mg/kg, completely eradicates the tumors (NMS249: 110-134%TGI vs. vc-MMAE: 114-143%TGI). Furthermore, a single 2 mg/kg dose of antiCD22-NMS249 causes three weeks of tumor stasis[1].

Nemorubicin (3'-deamino-3'-[2''(S)-methoxy-4''-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682 . Experimental design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively. Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. Conclusions: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.[1]

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Correlation Studies. [1]
MMDX (20 μmol/L) was incubated with microsomal fractions from 10 individual human livers; the incubation protocol was the same as that described above. The rates of PNU-159682 formation obtained in these experiments were correlated with several known CYP form-selective catalytic activities evaluated in the same microsomal samples (data provided by BD Gentest except those for nifedipine oxidation and erythromycin N-demethylation). Coefficients of determination (r2) and P values were determined by linear regression analysis.

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Chemical and Immunochemical Inhibition Studies. [1]
Formation of PNU-159682 from 20 μmol/L MMDX by pooled HLMs was evaluated in the absence (i.e., control) and presence of known CYP form-selective chemical inhibitors. The following inhibitors were examined at concentrations previously identified as being appropriate to cause CYP form-selective inhibition in HLMs: 7,8-benzoflavone (1 μmol/L, CYP1A2-selective), sulfaphenazole (20 μmol/L, CYP2C9-selective), quinidine (5 μmol/L, CYP2D6-selective), diethyldithiocarbamate (25 μmol/L; CYP2A6/E1-selective), troleandomycin (100 μmol/L, CYP3A-selective) and ketoconazole (1 μmol/L, CYP3A-selective). In experiments with reversible inhibitors, i.e., 7,8-benzoflavone, quinidine, sulfaphenazole, and ketoconazole, the inhibitor was coincubated with the substrate; the incubation protocol was the same as described above. In experiments with mechanism-based inhibitors, i.e., diethyldithiocarbamate and troleandomycin, the inhibitor was preincubated with liver microsomes and NADPH (0.5 mmol) at 37°C for 15 minutes before adding the substrate and additional 0.5 mmol NADPH. The reactions were then conducted as described above.
Immunochemical inhibition studies were carried out using mouse ascites fluids containing inhibitory MAbs which have been shown to be specific for different human CYP enzymes. Pooled HLMs (0.25 mg microsomal protein/mL; 20 pmol of total CYP) were preincubated with the designated amount of mouse ascites containing anti-CYP MAb (20-140 μg) at 37°C for 5 minutes in 0.3 mol/L Tris (pH 7.4); the reaction was then initiated by the addition of MMDX (final concentration, 20 μmol/L) and NADPH (final concentration, 0.5 mmol/L) in a total volume of 0.2 mL, and conducted as described above. The highest concentration of each MAb used in these trials (i.e., 7 μg ascites protein/pmol of total CYP) was previously shown to be saturating for an appropriate CYP form-specific reaction in HLMs. Control incubations were carried out in the absence of MAb.

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Incubation of MMDX with cDNA-expressed Human Cytochrome P450 Enzymes [1]
Incubations of MMDX with microsomes containing cDNA-expressed CYP enzymes were done as described for HLMs, except that the amount of enzyme used was 50 pmol/mL and incubations were terminated after 60 minutes; substrate concentration was 20 μmol/L. All incubations were done in duplicate. Aliquots of the supernatants from each sample were analyzed for PNU-159682 content by HPLC with fluorescence detection.

Cell Line: Jurkat, CEM, HT-29, A2780, DU145, and EM-2 cells
Concentration: 0-500 nM
Incubation Time: cultivated in compound-free medium for 72 hours after being exposed to PNU-159682 for one hour.
Result: was more potent than doxorubicin by 6,420 to 2,100 fold and MMDX by 2,360 to 790 fold, respectively.
PNU-159682's displayed IC70 values are in the subnanomolar range (0.07-0.58 nM) and significantly less than those found for doxorubicin and MMDX.

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In vitro Cytotoxicity [1]
The cytotoxic effects of doxorubicin, MMDX, and PNU-159682 on adherent tumor cell lines (HT-29, A2780, and DU 145) were evaluated using the sulforhodamine B assay as described by Skehan et al.; the effects of the drugs on the growth of nonadherent tumor cell lines (CEM, Jurkat, and EM-2) were evaluated by counting the surviving cells at the end of the treatment period with a ZM Cell Counter. Exponentially growing cells were seeded 24 hours before treatment and exposed to drugs for 1 hour, after which the medium was withdrawn and cells were incubated in a drug-free medium for 72 hours; control cells were not exposed to the drugs. Within each experiment, determinations were done in six times. IC70 values were then calculated from semilogarithmic concentration-response curves by linear interpolation. Data were expressed as mean ± SE of at least three independent experiments.

Animal Model: MX-1 tumor fragments in four- to six-week-old female CD-1 athymic nude mice[1]
Dosage: 4 μg/kg
Administration: Intravenous injection; q7dx3; 40 days
Result: demonstrated anti-cancer properties in human mammary carcinoma xenografts (MX-1) treated with PNU-159682 .

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Disseminated L1210 Leukemia. [1]
Eight-week-old inbred female CD2F1 (BALB/c × DBA/2) were used for evaluation of the therapeutic efficacy of PNU-159682 , in comparison with that of MMDX. Disseminated neoplasia was induced by i.v. injection of 105 L1210 cells; 1 day later, the animals were randomly assigned to an experimental group (n = 10) and received a single i.v. injection of MMDX, PNU-159682 , or saline (control group). Treatment efficacy was evaluated by comparing the median survival time in the treated and control groups, and expressed as increase in life span as follows: % increase in life span = (100 × median survival time of drug treated mice / median survival time of control mice) − 100. Statistical comparison between the groups was made using the nonparametric Mann-Whitney test.

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Subcutaneous MX-1 Human Mammary Adenocarcinoma Xenografts. [1]
Four- to six-week-old female CD-1 athymic nude mice (from Charles River) were used for evaluation of the activity of PNU-159682 against MX-1 human mammary carcinoma xenografts. On day 0, animals (n = 14) were grafted s.c. with MX-1 tumor fragments in the right flank. Eight days later, they were randomly assigned to the drug treatment group or control group (n = 7 mice per group), and treatment was started. PNU-159682 was given i.v. (4 μg/kg) according to a q7dx3 (every 7 days for three doses) schedule; control animals received saline injections. Tumor volume was estimated from measurements done with a caliper using the formula: tumor volume (mm3) = D × d2 / 2; where D and d are the longest and the shortest diameters, respectively. For ethical reasons, control animals were sacrificed on day 21 when the mean tumor volume in the group was ∼2,500 mm3; animals receiving drug treatment were monitored up to day 50, at which point they were sacrificed.

[1]. Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes. Clin Cancer Res. 2005 Feb 15;11(4):1608-17.

[2]. Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma. Mol Cancer Ther. 2016 Dec;15(12):2926-2935.

[3]. Recent advances of antibody drug conjugates for clinical applications. Acta Pharm Sin B. 2020 Sep;10(9):1589-1600.

[4]. Interim data: Phase I/IIa study of EGFR-targeted EDV nanocells carrying cytotoxic drug PNU-159682 (E-EDV-D682) with immunomodulatory adjuvant EDVs carrying α-galactosyl ceramide (EDV-GC) in patients with recurrent, metastatic pancreatic cancer. GASTROINTESTINAL CANCER—GASTROESOPHAGEAL, PANCREATIC, AND HEPATOBILIARY. Journal of Clinical Oncology, Volume 38, Number 15_supplMay 2020

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V(max) and intrinsic metabolic clearance (CL(int)) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL(int) and previously reported values of MMDX LD(50) for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation. Source: Biochem Pharmacol. 2008 Sep 15;76(6):784-95.

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Purpose: Nemorubicin (3'-deamino-3'-[2''(S)-methoxy-4''-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682. Experimental design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively. Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. Conclusions: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.[1]

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In most cases, cytotoxic drugs do not preferentially accumulate at the tumor site, causing unwanted toxicities and preventing dose escalation to therapeutically active regimens. Here, we show that acetazolamide derivatives, which bind to carbonic anhydrase IX (CAIX) on the surface of kidney cancer cells, selectively deliver payloads at the site of disease, sparing normal organs. Biodistribution studies, performed in tumor-bearing mice with acetazolamide derivatives bearing a technetium-99m chelator complex or a red fluorophore as payload, revealed a preferential tumor accumulation of the compound at doses up to 560 nmol/kg. The percentage of injected dose per gram in the tumor was dose-dependent and revealed optimal tumor:organ ratios at 140 nmol/kg, with a tumor:blood ratio of 80:1 at 6 hours. Acetazolamide, coupled to potent cytotoxic drugs via a dipeptide linker, exhibited a potent antitumor activity in nude mice bearing SKRC-52 renal cell carcinomas, whereas drug derivatives devoid of the acetazolamide moiety did not exhibit any detectable anticancer activity at the same doses. The observation of tumor regression with a noninternalizing ligand and with different cytotoxic moieties (MMAE and PNU-159682) indicates a general mechanism of action, based on the selective accumulation of the product on tumor cells, followed by the extracellular proteolytic release of the cytotoxic payload at the neoplastic site and the subsequent drug internalization into tumor cells. Acetazolamide-based drug conjugates may represent a promising class of targeted agents for the treatment of metastatic kidney cancer, as the majority of human clear cell renal cell carcinomas are strongly positive for CAIX. Mol Cancer Ther; 15(12); 2926-35.[2]

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Antibody drug conjugates (ADCs) normally compose of a humanized antibody and small molecular drug via a chemical linker. After decades of preclinical and clinical studies, a series of ADCs have been widely used for treating specific tumor types in the clinic such as brentuximab vedotin (Adcetris®) for relapsed Hodgkin's lymphoma and systemic anaplastic large cell lymphoma, gemtuzumab ozogamicin (Mylotarg®) for acute myeloid leukemia, ado-trastuzumab emtansine (Kadcyla®) for HER2-positive metastatic breast cancer, inotuzumab ozogamicin (Besponsa®) and most recently polatuzumab vedotin-piiq (Polivy®) for B cell malignancies. More than eighty ADCs have been investigated in different clinical stages from approximately six hundred clinical trials to date. This review summarizes the key elements of ADCs and highlights recent advances of ADCs, as well as important lessons learned from clinical data, and future directions.[3]

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Background: Targeted EDV nanocells loaded with doxorubicin and microRNA16a have shown excellent safety profiles in Phase I trials in recurrent glioma and mesothelioma. This planned safety analysis of an ongoing first-in-human, open label Phase I/IIa study in patients with treatment-refractory metastatic pancreatic cancer, assesses safety, biologic and clinical activity of EGFR-targeted EDV nanocells carrying cytotoxic drug PNU-159682, designed to overcome drug resistance, combined with EDV nanocells carrying immunomodulatory adjuvant α-galactosyl ceramide, designed to stimulate anti-tumour immune response. Methods: 9 patients with advanced pancreatic cancer enrolled in the dose escalation phase to evaluate safety of the EDV combination. Doses gradually escalated from 2 x 109 EDVs/dose to a maximum of 7 x 109 EDVs/dose in Week 7, with subsequent dosing at the maximum dose achieved in Cycle 1. iRECIST criteria was used to assess tumour response after each cycle, and blood was collected each cycle for cytokine and PBMC analysis. Results: Combination EDVs were well tolerated with no DLTs, and no drug related SAEs. A minority of patients experienced G1 infusion reactions, which responded promptly to supportive treatment. PR or SD was achieved at 8 weeks in 8/9 patients (CBR 89%), with responses confirmed at 4 months in 4/5 evaluable patients (80%), with 2 durable responses seen beyond 6 months. Exploratory analyses have revealed elevation of IFN-α and IFN-γ in almost all evaluable patients (6/8). In addition, we observed elevated CD8+ T cells (2/8), iNKT, dendritic and NK cells (3/8), and a reduction in exhausted CD8+ T cells (3/8), suggesting activation of both innate and adaptive immune responses. Conclusions: EDVs carrying the cytotoxic drug and immune adjuvant are safe and well tolerated. Early signals point to durable responses, possibly related to the development of an innate and adaptive immune response along with cytotoxic effects on drug resistant tumour cells. The Phase IIa study plans to enrol an additional 35 patients to further evaluate safety and anti-tumour efficacy. Clinical trial information: ACTRN12619000385145.[4]

C32H35NO13

641.6192

641.21

C, 61.24; H, 5.94; N, 2.23; O, 30.59

202350-68-3

PNU-159682;202350-68-3

9874188

Reddish Brown to red solid powder

1.6±0.1 g/cm3

838.5±65.0 °C at 760 mmHg

460.9±34.3 °C

0.0±3.2 mmHg at 25°C

1.691

6.18

4

14

6

46

1200

8

C[C@H]1[C@@H]2[C@H](C[C@@H](O1)O[C@H]3C[C@@](CC4=C3C(=C5C(=C4O)C(=O)C6=C(C5=O)C(=CC=C6)OC)O)(C(=O)CO)O)N7CCO[C@@H]([C@H]7O2)OC

SLURUCSFDHKXFR-WWMWMSKMSA-N

InChI=1S/C32H35NO13/c1-13-29-16(33-7-8-43-31(42-3)30(33)46-29)9-20(44-13)45-18-11-32(40,19(35)12-34)10-15-22(18)28(39)24-23(26(15)37)25(36)14-5-4-6-17(41-2)21(14)27(24)38/h4-6,13,16,18,20,29-31,34,37,39-40H,7-12H2,1-3H3/t13-,16-,18-,20-,29+,30+,31-,32-/m0/s1

(7S,9S)-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-7-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,9,10,12-tetrahydrotetracen-5(8H)-one

PNU-159682; PNU 159682; PNU159682； PNU-159682; 202350-68-3; UNII-CQ5A9ZNT7C; CQ5A9ZNT7C; (8S,10S)-6,8,11-Trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione; (8S,10S)-7,8,9,10-Tetrahydro-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-octahydro-9-methoxy-1-methyl-1H-pyrano(4',3':4,5)oxazolo(2,3-C)(1,4)oxazin-3-yl)oxy)-5,12-naphthacenedione;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~155.86 mM）

10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: ≥ 2.5 mg/mL (3.90 mM) (Please use freshly prepared in vivo formulations for optimal results.)